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Liao Y.,Novoprotein Scientific Inc. Shanghai | Chen S.,Novoprotein Scientific Inc. Shanghai | Wang D.,Novoprotein Scientific Inc. Shanghai | Zhang W.,Novoprotein Scientific Inc. Shanghai | And 7 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2013

Formaldehyde dehydrogenase (FDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family which oxidizes toxic formaldehyde to formate using NAD+ as an electron carrier. Three-dimensional structures have been reported for FDHs from several different species. Most FDHs are dependent on glutathione for catalysis, but the enzyme from Pseudomonas putida is an exception. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.7Å resolution of FDH from P. aeruginosa are described. Both the tetrameric assembly and the NAD+-binding mode of P. aeruginosa FDH are similar to those of P. putida FDH, which is in good agreement with the high sequence identity (87.97%) between these two proteins. Preliminary enzymatic kinetics studies of P. aeruginosa FDH also revealed a conserved glutathione-independent 'ping-pong' mechanism of formaldehyde oxidization. © 2013 International Union of Crystallography All rights reserved.

Zhang W.,Novoprotein Scientific Inc Shanghai | Zhang W.,Fudan University | Chen S.,Novoprotein Scientific Inc Shanghai | Liao Y.,Novoprotein Scientific Inc Shanghai | And 6 more authors.
Protein Expression and Purification | Year: 2013

As a member of zinc-containing medium-chain alcohol dehydrogenase family, formaldehyde dehydrogenase (FDH) can oxidize toxic formaldehyde to less active formate with NAD+ as a cofactor and exists in both prokaryotes and eukaryotes. Most FDHs are well known to be glutathione-dependent in the catalysis of formaldehyde oxidation, but the enzyme from Pseudomonas putida is an exception, which is independent of glutathione. To identify novel glutathione-independent FDHs from other bacterial strains and facilitate the corresponding structural and enzymatic studies, high-level soluble expression and efficient purification of these enzymes need to be achieved. Here, we present molecular cloning, expression, and purification of the FDH from Pseudomonas aeruginosa, which is a Gram-negative pathogenic bacterium causing opportunistic human infection. The FDH of P. aeruginosa shows high sequence identity (87.97%) with that of P. putida. Our results indicated that coexpression with molecular chaperones GroES, GroEL, and Tig has significantly attenuated inclusion body formation and improved the solubility of the recombinant FDH in Escherichia coli cells. A purification protocol including three chromatographic steps was also established to isolate the recombinant FDH to homogeneity with a yield of ∼3.2 mg from 1 L of cell culture. The recombinant P. aeruginosa FDH was properly folded and biologically functional, as demonstrated by the mass spectrometric, crystallographic, and enzymatic characterizations of the purified proteins. © 2013 Elsevier Inc. All rights reserved.

Novoprotein Scientific Shanghai Inc. | Date: 2010-08-12

The invention provides the protein mixture including the fusion proteins of C-myc, SOX2, KLF4, OCT-4, wherein each protein comprises a protein transduction domain (PTD) and a small ubiquitin-like modifier (SUMO) fused with the said protein. The invention further provides the preparation method of the protein mixture and its use for inducing human pluripotent stem cell.

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